The development of therapeutic antibodies represents a revolutionary era in the long history of human medicine. More than 30 antibodies have been approved for human therapy, and over 250 antibodies are in clinical development worldwide for a wide range of major diseases, including cancer, autoimmunity, inflammation, cardiovascular disease, infectious diseases and ocular disease. Over the past decade, the market for monoclonal antibody products has grown exponentially, propelled by the success of such blockbuster drugs as trastuzumab, bevacizumab, rituximab, infliximab and adalimumab. While these first-generation antibody therapeutics have benefited numerous patients, advances in antibody technology and a deeper understanding of the mechanism of action have paved the way for improved versions of antibodies with even better efficacy and fewer side effects.
Successful development and viable use of antibody therapeutics pose many unique challenges compared to traditional medicines that are small organic and inorganic molecules. The biophysical properties of antibodies, like all proteins, are important determinants of their behavior and have significant impacts for development of therapeutics relating to expression, purification, formulation, storage, delivery, pharmacokinetics, immunogenicity and dosing regimens. Among the many characteristics, protein stability is a main feature defining the quality of a candidate antibody and its desirability as a successful therapeutic.
Protein therapy often requires delivering high dose of the protein to patients in order to achieve the desired efficacy. Meanwhile, certain routes of administration are associated with limitations such as delivery time, volume and physical force that require the high dose protein to be in a high-concentration formulation (e.g., at least 100 mg/ml). However, highly concentrated protein formulations pose particular challenges with respect to stability, solubility, viscosity and other protein properties.
Proteins can be unstable and become degraded via multiple physical and chemical degradation pathways. Physical instability occurs mainly via two pathways—denaturation and aggregation, whereas chemical instability can occur via many pathways, such as deamidation, isomerization, cross-linking, oxidation, and fragmentation. Antibody instability is undesirable for drug development, as it can lead to decreased amount of active drug and lower in vivo efficacy, increased variability among batches of the therapeutics, and perhaps most importantly, immunogenicity in patients against aggregates and degradants. Wang et al (2007) J. Pharm. Sci. 96:1-26; Moore et al (1980) J Clin Endocrinology & Metabolism 51: 691-697; Rosenberg et al (2006) AAPSJ 8:E501-7; Joubert et al (2011) J Biol Chem 286: 25118-25133; Joubert et al (2012) J Biol Chem (2012) 286:25266-79).
Antibodies are large multidomain proteins, and factors contributing to their stability and propensity to aggregate are complex, including many extrinsic conditions such as temperature, pH, concentration, ionic strength and physical stress. Equally critical is the protein's own primary sequence. Although by nature the Fc region is largely identical between antibodies of a particular isotype, the Fab region differs greatly. Thus, there are significant variations in stability and aggregation propensity between antibodies, largely due to Fab sequence differences and the particular antigen specificity of the antibody. Lowe et al. (2011) Adv. Protein Chem. Struct Biol. 84:41-61.
The complement system plays a central role in the clearance of immune complexes and the immune response to infectious agents, foreign antigens, virus-infected cells and tumor cells. However, complement is also involved in pathological inflammation and in autoimmune diseases. Therefore, inhibition of excessive or uncontrolled activation of the complement cascade could provide clinical benefit to patients with such diseases and conditions.
The complement system encompasses three distinct activation pathways, designated the classical, mannose-binding lectin, and the alternative pathways. V. M. Holers In Clinical Immunology: Principles and Practice, ed. R. R. Rich, Mosby Press; 1996, 363-391. The classical pathway is a calcium/magnesium-dependent cascade which is normally activated by the formation of antigen-antibody complexes. The mannose-binding lectin (MBL) pathway is initiated by the binding of MBL to carbohydrate structures on pathogens, resulting in the activation of MBL protease (MASP) that cleaves C2 and C4 to form active C2a, C2b, C4a and C4b. The alternative pathway is a magnesium-dependent cascade which is activated by deposition and activation of C3 on certain susceptible surfaces (e.g., cell wall polysaccharides of yeast and bacteria, and certain biopolymer materials). Activation of the complement pathway generates biologically active fragments of complement proteins, e.g., C3a, C4a and C5a anaphylatoxins and C5b-9 membrane attack complexes (MAC), which mediate inflammatory activities involving leukocyte chemotaxis, activation of macrophages, neutrophils, platelets, mast cells and endothelial cells, vascular permeability, cytolysis, and tissue injury.
Factor D is a highly specific serine protease essential for activation of the alternative complement pathway. It cleaves factor B bound to C3b, generating the C3b/Bb enzyme which is the active component of the alternative pathway C3/C5 convertases. Factor D may be a suitable target for inhibition, since its plasma concentration in humans is very low (1.8 μg/ml), and it has been shown to be the limiting enzyme for activation of the alternative complement pathway. P. H. Lesavre and H. J. Müller-Eberhard. (1978) J. Exp. Med. 148: 1498-1510; J. E. Volanakis et al. (1985) New Eng. J. Med. 312: 395-401.
The down-regulation of complement activation has been demonstrated to be effective in treating several disease indications in animal models and in ex vivo studies, e.g., systemic lupus erythematosus and glomerulonephritis, rheumatoid arthritis, cardiopulmonary bypass and hemodialysis, hyperacute rejection in organ transplantation, myocardial infarction, reperfusion injury, and adult respiratory distress syndrome. In addition, other inflammatory conditions and autoimmune/immune complex diseases are also closely associated with complement activation, including thermal injury, severe asthma, anaphylactic shock, bowel inflammation, urticaria, angioedema, vasculitis, multiple sclerosis, myasthenia gravis, membranoproliferative glomerulonephritis, and Sjögren's syndrome.
Age-related macular degeneration (AMD) is a progressive chronic disease of the central retina with significant consequences for visual acuity. Lim et al. (2012) Lancet 379:1728. Late forms of the disease are the leading cause of vision loss in industrialized countries. For the Caucasian population ≥40 years of age the prevalence of early AMD is estimated at 6.8% and advanced AMD at 1.5%. de Jong (2006) N. Engl. J. Med. 355: 1474. The prevalence of late AMD increases dramatically with age rising to 11.8% after 80 years of age. Two types of AMD exist, non-exudative (dry) and exudative (wet) AMD. The more common dry form AMD involves atrophic and hypertrophic changes in the retinal pigment epithelium (RPE) underlying the central retina (macula) as well as deposits (drusen) on the RPE. Advanced dry AMD can result in significant retinal damage, including geographic atrophy (GA), with irreversible vision loss. Moreover, patients with dry AMD can progress to the wet form, in which abnormal blood vessels called choroidal neovascular membranes (CNVMs) develop under the retina, leak fluid and blood, and ultimately cause a blinding disciform scar in and under the retina.
Drugs targeting new blood vessel formation (neovasculazation) have been the mainstay for treating wet AMD. Ranibizumab, which is an anti-VEGFA antibody fragment, has proven to be highly effective in improving vision for patients afflicted with wet AMD. Recent studies have implicated an association between AMD and key proteins in the complement cascade and a number of therapies targeting specific complement components are being developed to treat dry AMD. A humanized anti-Factor D Fab fragment (aFD, lampalizumab; FCFD4514S) that potently inhibits Factor D and the alternative complement pathway, through binding to an exosite on factor D is currently in clinical development for the treatment of GA associated with dry AMD. Katschke et al. (2012) J Biol. Chem. 287:12886. A recent phase II clinical trial has shown that monthly intravitreal injection of lampalizumab effectively slowed the progression of GA lesions in patients with advanced dry AMD.
Eyes have many unique biophysical and anatomic features that make the ocular drug delivery more challenging. For example, blood-ocular barriers are defense mechanisms to protect the eye from infection, but at the same time make it hard for drug to penetrate, especially for diseases in the posterior segments of the eye. Consequently, high-dose administration is often desired to achieve and maintain drug's onsite bioavailability (e.g., ocular residence time) in order to improve efficacy. Meanwhile, the limited space in the back of the eye restrains the drug volume to be delivered, which in turn demands drugs to be delivered in a high concentration formulation.
Patients with ocular diseases can also benefit from long acting/slow released delivery of therapeutics. Less frequent dosing would provide improved convenience to the patient, have potential benefits of decreased infection rate and increased clinical efficacy. Controlled release of high dose drugs could also minimize drug side effects. Two promising systems for long-acting delivery are PLGA-based solid implants and an implantable port delivery system (PDS). Both systems have the potential to provide near zero-order release kinetics for an extended period of time. For PLGA implants the protein drug is encapsulated in a hydrophobic polymer matrix and drug release is accomplished via slow hydrolysis of the polymer. The rate of release can be controlled by changing the drug loading, polymer hydrophobicity, or polymer molecular weight. The PDS is a refillable device where release into the vitreous is controlled by a porous metal membrane comprising a titanium frit. Since the reservoir has a low volume, a high protein concentration is required for effective delivery with the PDS.
The conditions that a drug is exposed to vary depending on the delivery system used. For incorporation into solid PLGA implants, lyophilized or spray-dried drug is used. Implants are produced using a hot-melt extrusion process such that the drug is briefly exposed to temperatures approaching 90° C. Although the drug remains in solid state for the duration of release, degradation of PLGA may expose the drug to a low pH environment. In contrast, drug delivered with the PDS is maintained at high concentration in liquid state and exposed to vitreous which is characterized as a reducing environment at physiological ionic strength and pH.
In addition to or in lieu of high concentration and long acting delivery, increased bioavailability (e.g., ocular residence time) of the drug can be achieved, or facilitated, by post-translational modifications, wherein the protein drug is covalently conjugated with natural or synthetic polymers such as polysialylation, HESylation (conjugation with hydroxyethyl starch) and PEGylation. Chen et al (2011) Expert. Opin. Drug Deliv. 8:1221-36; Kontermann (2009) BioDrugs 23:93-109. PEGylation, the covalent attachment of polymer polyethylene glycol (PEG) to a protein, is a well-established technology especially useful for extending the half-life of antibody fragment therapeutics. Jevsevar et al. (2010) Biotech. J. 5:113-128.
Thus, there exists a great need for anti-factor D antibodies, as well as conjugates thereof, having improved stabilities, and that in some embodiments, are suitable for high concentration formulation and/or long acting delivery.